A variety of materials have been used for isolation and identification of biologically active substances, generally, and ligands particularly. As techniques have been developed for isolation of such biologically active substances, the ability to discriminate among the materials present in a mixture and selectively separate those desired has increased. One of the techniques which has been developed and refined to permit ever-increasing selectivity employs solid supports, especially particulate supports, on which a biologically active substance is ultimately bound and retained, either by a physical attraction, such as by adsorption, or by a chemical reaction. The significant interest in such a potent tool for immobilizing and either isolating and identifying biologically active substances or ligands or for use of such materials in subsequent procedures is at least partially attributable to the ease of manipulation of such solid supports or substrates and thereby the immobilized substances. The availability of such supports and procedures for immobilizing biological materials has stimulated research directed to improving and providing greater selectivity in techniques employed for separation or concentration of biologically active materials, such as affinity purification, particularly affinity chromatography, and the biospecific adsorbents used for such purposes, including solid supports, biologically active materials and, in some instances, activating or linking agents used to form a bond between a biologically active material and the solid support.
Those materials used as solid supports or carriers for binding biologically active materials have included most frequently polymers, usually of natural origin, containing hydroxyl groups in free or esterified form, such as agarose, cellulose, including cellulose esters such as cellulose nitrate, cellulose acetate, cellulose propionate, and the like, and acrylamide polymers and copolymers, such as polyacrylamide and acrylamide copolymer gels.
Many combinations of solid support and linking agent have been somewhat effective in immobilizing biologically active materials and specific compounds, either in removal of such materials from fluids, performing specific biological reactions or for use in immunoassays. Thus, particular choices of solid support and linking or activating compounds, when used to immobilize one member of an immune or biospecific complex such as in an antigen/antibody pair, hapten/antibody pair, apoprotein/cofactor pair, lectin/carbohydrate pair, or the like, have proven to be somewhat effective when the combination of solid support and the immobilized one member is used for binding an immune or biospecific ligand, that is, the second member of the biospecific or immune complex, to thereby form the immobilized complex.
The ability to immobilize a particular biologically active material, such as an antibody, particularly a monoclonal antibody, on a solid support provides an important tool in binding and qualitatively identifying and/or quantifying, or simply removing from a solution, the other member of an immune or biospecific complex, such as an antigen. However, the number of solid supports, linking agents or combinations of support and linking agent capable of immobilizing a wide variety of biologically active materials is limited.
Despite notable developments which have occurred in the technology related to immobilization of biologically active materials on solid supports, limitations in the techniques have frequently resulted from either the chemical or physical properties of the support materials employed.
Among the chemical shortcomings of materials used either as solid supports or as agents for coupling a biologically active material to the support have included:
(a) weak chemical bonding, PA1 (b) strong chemical bonding, thereby inhibiting attempts to elute a desired material from the column, PA1 (c) the necessity to employ somewhat extreme conditions to effect reaction, such as high temperature and pH, PA1 (d) coupling or activating agents which are capable of reacting with only a limited number of chemical groups on either a solid support or a biologically active compound, and PA1 (e) solid supports which have only a limited number of chemical groups capable of reacting with coupling or activating agents.
Many of the solid supports currently available exhibit mechanical properties which limit their usefulness in affinity chromotography applications. Conventional affinity chromotography materials generally employ biologically active materials immobilized on solid beads which are packed into tubular columns. The use of compressible materials, such as those from which most beads are made, which materials are currently preferred, may limit the dimensions and capacity of the column due to compression of the beads under excessive hydrostatic pressure resulting in an increase in pressure drop through the column and a subsequent decrease in flow rate, as well as a decrease in bead life. The efficiency of such columns may be further limited since a decrease in the size of the beads to provide an increase in surface area and, thereby, an increase in the effectiveness or rate of removal of material from the olution passing through the column, may cause this increase in pressure drop and decrease in flow rate to become more pronounced. In those situations in which recovery of biological material is performed on a large scale using large volumes of solution, as on a commercial preparative scale, the shortcomings of such beads become magnified. Thus, column dimensions must be selected as a compromise between elongated columns providing satisfactory separation but lacking reasonable flow rates and those allowing adequate flow rates but recovering biological material of reduced purity.
Although support materials in the form of membranes have been developed which overcome many of the aforementioned problems, particularly some of the chemical problems, many of these materials are asymmetric or skinned membranes which either require somewhat elaborate apparatus for their use or a means to provide a substantial head of pressure in order to effect flow of ligand-containing solutions through the asymmetric membranes.